Plasminogen, a 92,000 Mr glycoprotein present in blood plasma, is normally incorporated as a zymogen to blood clots where upon proteolytic activation and release of the 74 amino acid long N-terminal polypeptide it transforms into plasmin, a protease with high specificity towards fibrin. Plasmin is structured by a light chain of 25,000 Mr that contains the catalytic center joined to a heavy chain integrated by five highly homologous sections, known as kringles (K) of ca. 10,000 Mr each. The kringles are presumed to be responsible for fibrin recognition and attachment to the clot and for binding of Alpha2-antiplasmin (an inhibitor) and streptokinase (an activator). The heavy chain also carries at least five binding sites for lysine and Omega-amino acid analogs that act as antifibrinolytics. We propose to investigate the conformation of human plasmogen in solution by NMR spectroscopy in order to characterize the binding sites of fragments K1, K4, K1+2+3, and K5 + light chain (miniplasminogen). A number of cyclic and linear antifibrinolytics that vary in their aromatic character will be studied. Accurate measurements of binding affinities and on/off rates will be implemented under steady state conditions and using double resonance techniques. The proposed allosterism between the N-terminal peptide and lysine for binding to K1 (2 site model) will be tested using a short synthetic peptide to simulate the effector. Spin decoupling, Overhauser enhancements, laser photo-CIDNP, chemical modification and 2D-NMR will be exploited to assign resonances, characterize the solution structures and follow conformational transitions. Such studies are expected: (a) to assist in the design of chemotherapies for vascular thrombosis and pulmonary embolism, and (b) to help understanding the mode of action of antifibrinolytic drugs used clinically in the treatment of haemorragic disorders.